1. Field of the Invention
The present invention relates to a method of detecting inorganic phosphoric acids or pyrophosphates. Further, the present invention relates to a method of simply detecting a targeted nucleic acid in a sample with high sensitivity through the detection described above, and in particular, a method for discriminating a SNP.
2. Description of the Related Art
In recent years, techniques relating to gene information have been actively developed. In a medical field, therapies of diseases at the molecular level have been enabled by analyses of a gene relating to the disease. Further, tailor made medical cares corresponding to every patient has been also enabled by gene diagnosis. In a pharmaceutical field, protein molecules such as antibodies and hormones have been specified using gene information, and utilized as a medicament. Also in agricultural or food fields, many products in which gene information is utilized have been manufactured.
Among such gene information, genetic polymorphisms are particularly important. Similarly to variations of our faces and body types, considerable part of the gene information also varies each person to person. Among the differences in such gene information, an alteration of the base sequence that is present in a frequency of 1% or greater of the population is referred to as a genetic polymorphism. Such genetic polymorphism is mentioned as relating to causes of various diseases, constitution, drug responsiveness, adverse reactions by drugs, and the like as well as facial appearance of each person. Currently, relationships between the genetic polymorphisms and diseases have been rapidly investigated.
Among the genetic polymorphisms, SNP (Single Nucleotide Polymorphism) has been particularly attracted the attention in recent years. SNP refers to a genetic polymorphism involving a difference in single base alone in the base sequence of gene information. SNP is referred to as existing in human genomic DNA by 2 to 3 million, and is readily utilized as a marker of a genetic polymorphism. Thus, the application thereof to the clinical field has been expected. At present, as related techniques to SNPs, development of typing techniques of SNPs in which a base is discriminated of the SNP site has been performed, in addition to studies on identification of a SNP site in a genome and on relationships between SNPs and diseases, and the like.
In general, as a means to discriminate the difference in a base sequence of a DNA, Sanger's (dideoxy) method, DNA tip method or the like has been used. In Sanger's method, SNP typing is executed through the amplification of a region to be analyzed of a human gene by a PCR (polymerase chain reaction) method followed by sequencing through reading the base sequence of a desired position to directly determine the base sequence. In the DNA tip method, a PCR product of a cDNA or an oligonucleotide probe having 15 to 20 bases is immobilized on a substrate, and thereto is hybridized a sample DNA which had been labeled with fluorescence. By strictly controlling the hybridization condition such as temperature and the like, SNP is detected on the basis of the difference of affinity between a probe and the sample DNA, i.e., by the difference in fluorescent intensities.
Moreover, in an attempt to allow for large scale SNP typing, JP-T (the term “JP-T” as used herein means a published Japanese translation of PCT Patent Application) No. 10-510982 (TaqMan PCR method) and JP-T No. 2001-518805 (Invader method) and the like have been disclosed. Diagrammic illustration of the method disclosed in JP-T No. 10-510982 is shown in FIG. 1. In JP-T No. 10-510982, an oligonucleotide probe is provided which includes a fluorescent molecule and a quencher molecule capable of quenching the fluorescence of the fluorescent molecule. An oligonucleotide probe is used which includes a reporter molecule-quencher molecule pair which is to be specifically annealed in a “downstream region” (i.e., in an extending direction of the primer binding site) of a target polynucleotide. These quencher molecule and reporter molecule are arranged close enough with each other. Accordingly, when the reporter molecule is thereby excited, this excitation state energy is constantly transmitted to the quencher molecule in a nonradioactive fashion. Thus, the energy is quenched in a nonradioactive fashion, or emitted at the frequency which is different from the luminescent frequency of the reporter molecule. The probe is annealed with a template during the chain extension by DNA polymerase, and this probe is then digested by 5′→3′ exonuclease of the polymerase. As a consequence of digestion of the probe, this reporter molecule is effectively separated from the quencher molecule. Accordingly, the quencher molecule can no longer get close enough to the reporter molecule such that it can quench the fluorescence of the reporter molecule. Therefore, the more the probe is digested upon amplification, number of the reporter molecules in the solution is increased, resulting in increase of number of nonquenching reporter molecules, thereby the fluorescent signal increasingly enhanced.
JP-T No. 2001-518805 discloses a method of detecting the presence of a target nucleic acid molecule. Diagrammic illustration of this method is shown in FIG. 2. A method is disclosed which comprises providing: a structure-specific nuclease, a source of a first target nucleic acid, said first target nucleic acid having a first region, a second region and a third region, wherein said first region is located adjacent to and downstream from said second region and wherein said second region is located adjacent to and downstream from said third region; a first oligonucleotide having a 5′ portion and a 3′ portion wherein said 5′ portion of said first oligonucleotide contains a sequence complementary to said second region of said first target nucleic acid and wherein said 3′ portion of said first oligonucleotide contains a sequence complementary to said third region of said first target nucleic acid; a second oligonucleotide having a 5′ portion and a 3′ portion wherein said 5′ portion of said second oligonucleotide contains a sequence complementary to said first region of said first target nucleic acid and wherein said 3′ portion of said second oligonucleotide contains a sequence complementary to said second region of said first target nucleic acid; a source of a second target nucleic acid, said second target nucleic acid having a first region, a second region and a third region, wherein said first region is located adjacent to and downstream from said second region and wherein said second region is located adjacent to and downstream from said third region; a third oligonucleotide having a 5′ portion and a 3′ portion wherein said 5′ portion of said third oligonucleotide contains a sequence complementary to said second region of said second target nucleic acid and wherein said 3′ portion of said third oligonucleotide contains a sequence complementary to said third region of said second target nucleic acid; generating a first cleavage structure wherein at least said 3′ portion of said first oligonucleotide is annealed to said first target nucleic acid and wherein at least 5′ portion of said second oligonucleotide is annealed to said first target nucleic acid and wherein cleavage of said first cleavage structure occurs via said structure-specific nuclease thereby cleaving said first oligonucleotide to generate a fourth oligonucleotide, said fourth oligonucleotide having a 5′ portion and a 3′ portion wherein said 5′ portion of said fourth oligonucleotide contains a sequence complementary to said first region of said second target nucleic acid and wherein said 3′ portion of said fourth oligonucleotide contains a sequence complementary to said second region of said second target nucleic acid; generating a second cleavage structure wherein at least said 3′ portion of said third oligonucleotide is annealed to said second target nucleic acid and wherein at least said 5′ portion of said fourth oligonucleotide is annealed to said second target nucleic acid and wherein cleavage of said second cleavage structure occurs to generate a fifth oligonucleotide, said fifth oligonucleotide having a 3′ hydroxyl group; and detecting said fifth oligonucleotide.
However, any of these methods uses fluorescent labeling for the probe, therefore, reagents and the like become very expensive, and a light source for laser irradiation or the like is required for detecting fluorescence, leading to size enlargement of the equipment for the measurement. Accordingly, there still exist many problems taking into account of the use for clinical applications in medical institutions.
To the contrary, as a new method of determining a DNA base sequence, JP-T No. 2001-506864 discloses a method in which a pyrophosphate, which is generated concurrent with an extension reaction of a DNA is converted into ATP (adenosine triphosphate), and luminescence by luciferin is detected on the basis of the action of luciferase using the ATP as a substrate. Diagrammic illustration of this method is shown in FIG. 3. Provided is method wherein an extension primer, which hybridizes to a sample DNA immediately adjacent to the target position is provided and the sample DNA and extension primer are subjected to a polymerase reaction in the presence of a deoxynucleotide or dideoxynucleotide whereby the deoxynucleotide or dideoxynucleotide will only become incorporated and release a pyrophosphate (PPi) if it is complementary to the base in the target position, any release of the PPi being detected enzymatically, different deoxynucleotides or dideoxynucleotides being added either to separate aliquouts of sample-primer mixture or successively to an aliquot of the same sample-primer mixture and subjected to the polymerase reaction to indicate which deoxynucleotide or dideoxynucleotide is incorporated, characterized in that, a PPi-detecting enzyme is included during the polymerase reaction step, and that deoxyadenosine triphosphate (dATP) or a dATP analog is used capable of acting as a substrate for polymerase but not capable of acting as a substrate for said PPi-detecting enzyme, instead of dATP or dideoxyadenosine triphosphate (ddATP). In JP-T No. 2001-506864, there is disclosed a method which advantageously permits a large scale unelectrophoretic solid phase DNA sequencing, thereby making successive determination of time-dependent progress of a polymerization reaction possible.
However, also in this method, a large scale equipment is required because detection by luminescence with luciferase and the like is required, which was not suitable as a simplified system for use in bed side at a hospital or use in drug supply.
In JP-A No. 63-49100, a method of measuring an inorganic phosphoric acid is disclosed. According to this method, a reaction including three steps involving an inorganic phosphoric acid is utilized, and finally, oxidized nicotinamide adenine dinucleotide (NAD+) is reduced to give NADH, or oxidized nicotinamide adenine dinucleotide phosphate (NADP+) is reduced to give NADPH. Amount of thus resulting NAD(P)H is measured by ultraviolet absorption method or the like. Because a reaction including three steps is used in this method, many elementary steps of the reaction are involved. Therefore, it is complicated to establish the reaction condition for allowing the entire elementary steps proceed. Further, it is not necessarily possible to predetermine an efficient condition. Moreover, it is required that comparatively large-scale equipment for the measurement such as spectrophotometer is used. Thus, there also existed problems involving lack of simpleness.
Moreover, similarly to the method described in the above JP-A No. 63-49100, a method of detecting a targeted sample is described in International Patent Publication pamphlet WO 00/04378 utilizing the reduction of a coenzyme NAD(P)+ concurrent with a reaction of the targeted sample. However, since the targeted sample for detection therein is L-phenylalanine, such a method is not applicable to the detection of presence of a target nucleic acid molecule.